Bacterial-excreted small volatile molecule 2-aminoacetophenone induces oxidative stress and apoptosis in murine skeletal muscle

Abstract

Oxidative stress induces mitochondrial dysfunction and facilitates apoptosis, tissue damage or metabolic alterations following infection. We have previously discovered that the Pseudomonas aeruginosa (PA) quorum sensing (QS)-excreted small volatile molecule, 2-aminoacetophenone (2-AA), which is produced in infected human tissue, promotes bacterial phenotypes that favor chronic infection, while also dampening the pathogen‑induced innate immune response, thus compromising muscle function and promoting host tolerance to infection. In this study, murine whole-genome expression data have demonstrated that 2-AA affects the expression of genes involved in reactive oxygen species (ROS) homeostasis, thus producing an oxidative stress signature in skeletal muscle. The results of the present study demonstrated that the expression levels of genes involved in apoptosis signaling pathways were upregulated in the skeletal muscle of 2-AA-treated mice. To confirm the results of our transcriptome analysis, we used a novel high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) method and observed increased levels of bisallylic methylene fatty acyl protons and vinyl protons, suggesting that 2-AA induces skeletal muscle cell apoptosis. This effect was corroborated by our results demonstrating the downregulation of mitochondrial membrane potential in vivo in response to 2-AA. The findings of the present study indicate that the bacterial infochemical, 2-AA, disrupts mitochondrial functions by inducing oxidative stress and apoptosis signaling and likely promotes skeletal muscle dysfunction, which may favor chronic/persistent infection.

Figure 1

2-Aminoacetophenone (2-AA) affects reactive oxygen species (ROS) metabolism and the response to oxidative stress in murine skeletal muscle. Black bars indicate the number of downregulated genes; gray bars indicate the number of upregulated genes in the skeletal muscle of mice 4 days after 2-AA treatment versus the control mice (left vertical axis). The negative log10 of p-values represented by gray triangles are indicated in the right vertical axis.

Figure 2

2-Aminoacetophenone (2-AA) affects genes involved in the apoptosis pathway in murine skeletal muscle. Black bars indicate the number of downregulated genes; gray bars indicate the number of upregulated genes in the skeletal muscle of mice 4 days after 2-AA treatment versus the control mice (left vertical axis). The negative log10 of p-values represented by gray triangles are indicated in the right vertical axis.

Figure 3

Nuclear magnetic resonance (NMR) spectra from ¹H-NMR high-resolution magic-angle-spinning (HRMAS) experiments performed on the gastrocnemius skeletal muscle specimens of mice. The spectra were acquired from normal and 2-aminoacetophenone (2-AA)-treated mice at 4 days post-2-AA treatment and scaled to the phosphocreatine and creatine peak (3.02 ppm). The lipid peak at 1.3 ppm is attributed to methylene protons of intra-myocellular triglyceride acyl chains, primarily due to intramyocellular lipids (IMCLs). Resonance signals are due to residual water (4.7–4.8 ppm); terminal methyl (0.8–1.0 ppm); acyl chain methylene (1.1–1.5 ppm); α- and β- methylene (2.0–2.5 ppm) and olefinic protons (5.4 ppm) of lipids; N-methyl protons of phosphocreatine and creatine (3.0 ppm); and N-trimethyl protons of betaines (3.2 ppm), which correspond to taurine and choline-containing compounds. Bisallylic methylene fatty acyl protons at 2.8 ppm correspond to polyunsaturated fatty acids (PUFAs), which accumulate due to apoptosis. Vinyl proton accumulation at 5.4 ppm, including protons from ceramide and possibly other sphingolipids suggests apoptosis.

Figure 4

2-Aminoacetophenone (2-AA) reduces mitochondrial membrane potential in murine skeletal muscle. Mitochondrial membrane potential was analyzed by in vivo microscopy. (A) In vivo fluorescence microscopic images are shown both for 2-AA-treated (left column) and untreated (right column) groups. Green fluorescence (top panel) from 3,3′-dihexyloxacarbocyanine iodide (DiOC₆) staining represents mitochondrial membrane potential, and red signal (bottom panel) from CellTracker Orange staining is for internal control staining. The white scale bar at the bottom represents 200 μm. (B) The average fluorescence signal was quantified by densitometry and shown as a bar graph with the standard error of the means. 2-AA-treated group showed a significantly decreased signal as compared to the controls. ^*p<0.05, according to Student's t-test.

Figure 5

Representative schematic diagram showing that 2-aminoacetophenone (2-AA) induces oxidative stress and apoptosis in skeletal muscle. 2-AA induces oxidative stress by generating reactive oxygen species (ROS). The oxidative damage and ROS reduce mitochondrial membrane potential (ΔΨ), and release Bcl-2 and cytochrome c, which promotes apoptosis. Lipid accumulation following 2-AA treatment potentially generates apoptotic signals in the cells (58,59). The red arrows denote the induction of the cellular components.